Mass spectrometry is used to identify unknown compounds, to qualify known materials, and to clarify the structural and chemical properties of molecules. Mass spectrometers can accomplish these functions with very minute quantities of sample material, e.g., less than picogram amounts, and at very low concentrations in chemically complex mixtures, e.g., one part in a trillion.
Mass spectrometry has been used, inter alia, to detect and identify steroid use by athletes; for real time breath monitoring of patients by anesthesiologists during surgery; to determine the composition of molecular species found in space; to locate oil deposits by measuring petroleum precursors in rock; to detect dioxins in contaminated fish; and to determine gene damage from environmental causes.
FIG. 1 shows a conventional mass spectrometer which includes an ionization source 1, an ion analyzer 2, an ion detector 3 and a data system 4. A sample is provided to the ionization source 1 via an inlet. The sample may be a solid, liquid, or vapor. However, to perform efficiently the functions of ionization, ion analysis and ion detection, the mass spectrometer usually requires the sample to be a gas, and the ionization source 1 to be a vacuum chamber. When introducing pure solids into the ionization source 1, the sample is simply placed on the tip of a rod that is inserted into the vacuum chamber 1 through a vacuum-tight seal. The introduced sample is then evaporated or sublimed into the gas phase, usually by applying heat. Gases and liquids can be introduced into the ionization source 1 through inlets with controlled flow.
After ionization, the ions are sorted in the mass analyzer 2 according to their mass-to-charge (m/z) ratios, and then are collected by the ion detector 3. In the ion detector 3, the ions generate an electrical signal that is proportional to the number of detected ions. The generated electrical signals are supplied to the data system 4 which records these electrical signals and then converts them into a mass spectrum, i.e., a graph of ion abundance vs. mass-to-charge ratio. The ions and their respective abundances serve to establish the molecular weight and structure of the compound being mass analyzed.
FIG. 2 shows the ionization source 1 implemented by a technique known as Electron Ionization (EI). In this technique, ions are generated in the ionization source 1 by bombarding the introduced gaseous molecules (sample) with a beam of electrons from a filament 5 at e.g., 70 eV. Since the energy of the bombarding electrons is much greater than the bonds which hold the molecule together, the high energy electrons interact with the gaseous molecule sample. As a result, ionization occurs, bonds are broken and ion fragments are formed. In the resulting mixture, positive ions are propelled into the analyzer 2 by applying a voltage on the repeller 6, and focused by voltages applied to the lens system 7. Negative ions and electrons are attracted to the positively charged cathode or electron trap 8. Finally, the resulting neutral molecules and fragments that are not ionized are pumped away.
While EI is commonly used for those molecules that can be vaporized, electron ionization with electrons accelerated through a potential of 70 volts is a highly energetic or "hard" process which may lead to extensive fragmentation that leaves very little or no trace of a molecular ion. In the absence of a molecular ion, molecular weight and structure are not easily determined. Further, relatively large molecules, such as complex proteins, require a significant amount of energy to induce fragmentation using EI. These problems have led to the development of lower energy ("soft") ionization techniques.
One such lower energy ionization technique is known as Chemical Ionization ("CI"). In contrast to electron ionization, CI produces ions by a relatively gentle process of proton transfer from an ionized reagent gas such as methane. Abundant protonated molecules generally result.
For example, the mass spectrum of ephedrine shows a fragment ion at m/z 58 and no molecular ion at m/z 165 under electron ionization conditions. However, under chemical ionization conditions, the mass spectrum shows a fragment ion at m/z 58 as in the EI, shows an abundant protonated fragment ion at m/z 148 which corresponds to a loss of water (18 mass units), and also shows an abundant protonated molecule at m/z 166. Thus, using chemical ionization, the fragment ion at m/z 58 and the molecular ion at m/z 166 can be detected.
As shown in FIG. 1, the ion analyzer 2 is coupled to the output of ionization source 1. In general, the ion analyzer serves to sort the ions from ionization source 1 according to their mass-to-charge ratios or a related property. Presently, there are three widely-used ion analyzers, namely, magnetic and electric sectors, quadrupoles, and ion traps.
Sector mass spectrometers use combinations of magnetic and electric fields to sort the ions. A common configuration for a sector instrument is the so-called Nier-Johnson geometry. In this sector instrument, ions of larger mass have trajectories of larger radius than ions of smaller mass. Ions of different mass-to-charge ratio are focused at a detector by varying the magnetic strength. This combination of electrostatic and magnetic analyzers provide mass resolution high enough to resolve ions of the same nominal mass, but different chemical formula, such as N.sub.2 and CO at m/z 28.
Another type of mass analyzer is the so-called quadrupole mass filter and consists of four poles or rods. In this device, mass sorting depends on ion motion resulting from dc and radio frequency (rf) electric fields. Quadrupole mass spectrometers provide lower resolution than do sector mass spectrometers, but are more easily interfaced to various inlet systems.
The ion trap mass spectrometer operates on a principle similar to the quadrupole mass spectrometer. However, rather than allowing ions to pass therethrough, the ion trap is able to store all ions for subsequent experiments. The ion trap mass spectrometer uses a field that is generated by a sandwich geometry including a ring electrode in the middle and caps on each end. The ion trap serves to trap ions of a selected range of mass-to-charge ratios in the space bound by the electrodes. A mass spectrum is produced by varying the electric field to eject sequentially ions of increasing mass-to-charge ratio for detection.
In the past, mass spectrometric methods were restricted to relatively small, volatile molecules. However, with the development of ionization methods including Fast Atom Bombardment (FAB), plasma desorption and laser desorption, mass analysis can now be conducted on large and volatile molecules. Specifically, these techniques have been successful in the production of intact large molecular ions. Accordingly, much effort has been employed to induce the fragmentation of these large molecular ions so as to improve the detailed information necessary for characterizing their molecular structures.
The primary means for accomplishing this task has been the introduction of tandem mass spectrometers or Mass Spectrometry/Mass Spectrometry (MS/MS) in which the molecular ion is mass selected by a first mass spectrometer (MS1), and then the selected molecular ion is fragmented with the resulting products analyzed by a second mass spectrometer (MS2) which is scanned to produce a product ion spectrum. The major advantage of MS/MS analysis is the ability to select the molecular ion of a single analyte from a mixture and obtain its mass spectrum.
When Fast Atom Bombardment (FAB) is used as the ionization technique, the normal mass spectra are characterized by an abundance of peaks arising from the liquid matrix, adduct ions, and a general peak-to-every-mass background. In many cases, several molecular ion species are produced. Selection of a single molecular ion species in an MS/MS scheme produces a mass spectrum whose peaks are unambiguously attributable to the analyte and for which the S/N ratio relative to the background ions is considerably improved.
FIG. 3 shows the basic scheme for a tandem mass spectrometer or MS/MS system. In the most common arrangement, a collision chamber containing an inert gas (usually helium) is placed between the two mass spectrometers. Ions emerging from MS1 collide with neutral inert gas atoms and are fragmented by a process known as collision induced dissociation (CID). These fragment (or product) ions are then mass analyzed by MS2. Because this process (CID) becomes less effective for large molecular ions, several alternative schemes have been developed including surface induced dissociation (SID), electron induced dissociation (EID) and photodissociation.
An additional, but not widely, used technique is known as neutralization/reionization mass spectrometry (NRMS). The basic scheme for this technique, utilized on a tandem mass spectrometer or MS/MS system, is shown in FIG. 4.
In this case, a mass-selected high energy ion beam from a first mass spectrometer MS1 is neutralized by collisions with a metal vapor in a first collision cell C1, and then reionized through collisions with a second reagent gas, such as O.sub.2, in a second collision chamber C2. The reionized molecule is then supplied to the second mass spectrometer for mass analysis.
In the system shown in FIG. 4, collisions with the vaporized metals in the first collision cell C1 favor charge exchange over collision-induced dissociation (CID). Reionization by collisions with O.sub.2 occurs in the second chamber, produces fragment ions. Both charge transfer processes, i.e., neutralization and reionization, take place at high kinetic energy and involve electron transfer. In the first collision cell C1, an electron is transferred from the metal vapor, such as a Hg vapor, to the primary molecule, thereby neutralizing the primary molecule. The thus neutralized primary molecule is then supplied to the second collision cell C2 in which an electron transfer process again occurs, this time from the neutralized molecule to the O.sub.2 vapor, thereby reionizing the primary molecule and at the same time producing fragment ions. The resulting ions are supplied from collision cell C2 to the second mass spectrometer for analysis by scanning in the B/E mode or with an electric sector (kinetic energy analyzer).
In FIG. 4, each of the first C1 and second C2 collision cells is a low pressure cell operating at a pressure such that the reagent gas is maintained at a pressure between 1 and 5 mTorr. Both processes, i.e., neutralization and reionization, take place at a high kinetic energy of approximately 4-5 keV.
A neutralization/reionization scheme involving charge exchange reactions has several disadvantages that are addressed by the proposed invention. Specifically, the system is relatively complex requiring two separate collision cells each employing a different reagent gas. The system also requires the neutralization and reionization processes to take place at relatively high kinetic energies. It is difficult to achieve such energies with large mass molecules. Finally, it does not address the fact that molecular ions produced by fast atom bombardment and other "soft" ionization techniques are generally even-electron protonated species that could more easily be neutralized by deprotonation.